Metakaryocidal Treatments

ABSTRACT

The invention provides, inter alia, methods of treating a disorder characterized by excessive metakaryotic stem cell growth by a combination metakaryocidal therapy. Also encompassed by the present invention are preventative methods comprising the administration of a metakaryocidal or metakayrostatic therapeutic agent.

RELATED APPLICATION(S)

This application is a Continuation of U.S. patent application Ser. No.16/430,768, filed on Jun. 4, 2019, which is a Continuation of U.S.patent application Ser. No. 15/126,369, filed on Sep. 15, 2016, which isa U.S. National Phase of International Application No.PCT/US2015/020988, filed on Mar. 17, 2015, which claims the benefitunder 35 U.S.C. § 119(e) of U.S. Provisional Application No. 61/954,426,filed on Mar. 17, 2014. The entire teachings of the above applicationsare incorporated herein by reference.

BACKGROUND OF THE INVENTION

After the early embryonic stages of development and plants and animals,metakaryotic stem cells arise from metamorphoses of embryonic stemcells. They drive the growth and development of organs up to maturityand in animals have been found to persist in a quiescent state “on call”to serve as the stem cells of wound healing. However, disorders such ascancers, vascular diseases, post-surgical restenoses and scleroderma aredriven by the abnormal growth and proliferation of this unique cell typeknown as metakaryotes or metakaryotic stem cells. Given thepervasiveness of these disorders, their immense social and financialcosts to society, the dearth of effective treatment for these disorders,and the peculiar biology of metakaryotic stem cells (which, at least inpart, is the reason for the ultimate ineffectiveness of existingtreatments), a need exists for effective treatments for disorderscharacterized by excessive metakaryotic stem cell growth and, of greaterlong term public health importance, to prevent such disorders.

SUMMARY OF THE INVENTION

The present invention covers methods of treating and preventingdisorders characterized by excessive metakaryotic stem cell growth in amammalian subject in need thereof comprising providing a therapeuticallyeffective metakaryocidal treatment, and in particular a combinationmetakaryocidal therapy, in which exposure of the subject to one or moreseparate metakaryocidal drugs is continuous and uninterrupted for atime/duration and at a dosage sufficient to achieve a bloodconcentration (i.e., therapeutic concentration) to effectively kill, orsignificantly inhibit the growth of metakaryotic stem cells therebytreating, or preventing, the disorder. The methods described herein canalso include the administration of concurrent or sequential eukaryocidaltreatment which effectively kills eukaryotic cells derived in part fromdifferentiation of the metakaryotic cells growing in the pathogeniclesion in the subject.

In one embodiment, an effective metakaryocidal treatment method, asdescribed herein, can comprise the administration of a singletherapeutically effective amount of a metakaryocidal agent. Candidatemetakaryocidal/metakaryostatic agents can be evaluated for theirtherapeutic efficacy using the methods described in InternationalApplication Number PCT/US15/20933, the teachings of which are herebyincorporated herein in their entirety.

As described herein, a therapeutically effective amount ofmetakaryocidal agent is an amount sufficient so as to achieve plasmaconcentrations of the agent for an uninterrupted duration substantiallysimilar to the in vitro metakaryocidal concentrations found to bemetakaryocidal in HT-29 or CAPAN-1 cell cultures for uninterrupteddurations sufficient to kill >99% of metakaryotic stem cells within theprecursor lesion(s) of the disorder, e.g. an adenomatous polyp of thehuman colon, a precursor of lethal adenocarcinomas and metastases asdescribed in the above-referenced application.

In another embodiment of the present invention, an effective combinationmetakaryocidal therapy comprises concurrent or sequential administrationof two, three, four, five or more courses of effective metakaryocidalagents. Importantly, the administration of the metakaryocides (andeukaryocides if applicable) comprises administering two or moremetakaryocidal agents so as to achieve plasma concentrations of theagents for an uninterrupted duration substantially similar to the invitro metakaryocidal concentrations of the agents found to bemetakaryocidal in HT-29 or CAPAN-1 cell cultures, as described above.That is, the effective course of metakaryocidal treatment for eachsingle agent/drug, or combination of agents/drugs is of a sufficientdosage of, and uninterrupted duration of time sufficient to killmetakaryotes associated with excessive metakaryotic stem cell growth.

However, although the translation of effective treatment from a cellculture to a human subject or other animal subject will be based on thedrug concentration effective in cell culture, but will not necessarilybe of the duration of uninterrupted treatment effective in a cellculture. Those skilled in the art, and as described in InternationalApplication Number PCT/US15/20933, will understand that metakaryoticstem cells may divide symmetrically and/or asymmetrically every day incell culture but at much longer intervals in pathogenic lesions such astumors or metastases, e.g. every twelve days in early human colonicadenocarcinomas. However, it is reasonable to believe that the durationof treatment effective in cell culture is translated to an effectivetreatment in mammals or humans when the duration of uninterruptedtreatment in mammals is substantially equivalent to the number ofsymmetrical plus asymmetrical metakaryotic cell divisions found to beeffective in cell culture. Thus, for example, a treatment with a singlemetakaryocide found effective for an uninterrupted duration of five daysin a cell culture comprising sequential symmetric plus asymmetricdivisions of metakaryotes in culture can reasonably require exposure ofmetakaryotes in a subject for a duration of up to weeks or months (e.g.,about 60 or more days to achieve an exposure period of five intervalsbetween symmetric plus asymmetric divisions of 12 days per interval) tobe effective in a human colonic adenocarcinoma.

The methods of the present invention encompass an effectivemetakaryocidal therapy wherein the metakaryocidal drugs contemplated tobe administered to the subject comprise an effective amount of anysingle, or combination of two or more agents such as acetaminophen,aspirin, captopril, celecoxib, daunorubicin, doxycycline, glyburide,metformin, verapamil or other NSAIDs, antibiotics or other agentsidentified as metakaryocidal to metakaryotic stem cells in culture orexplants derived from lesions of mammalian/human disorders. Inparticular, based on observations of two weeks exposure of HT-29 cellsin cell culture, the methods comprise administering metakaryocides at aconcentration and for a time sufficient for an effective therapy whereinthe amount of captopril achieves a plasma concentration of about 0.125to about 0.5 μM (e.g., about 0.25 μM), the effective amount of celecoxibachieves a plasma concentration of about 12.5 to about 50 μM (e.g.,about 25 μM), the effective amount of doxycycline achieves a plasmaconcentration of about 2.5 to about 10 μM (e.g., about 5 μM), theeffective amount of metformin achieves a plasma concentration of about100 to about 800 μM (e.g., about 200 μM), and the effective amount ofverapamil achieves a plasma concentration of about 2 to about 8 μM(e.g., about 4.0 μM). The metakaryocidal treatments described herein assingle or combination metakaryocidal therapy are each administered for aperiod of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 weeks. Thecombination therapy can further encompass a resting interval of 1, 2, 3or more days, or 1 or 2 or more weeks or months between theadministration of the metakaryocidal agents. Moreover, these drugs atconcentrations indicated can be effective for the prevention (i.e.,decreased likelihood) of diseases in which metakaryotes serve as thestem cells of precursor lesions when used singly over a sufficientuninterrupted period that kills at least 99% of the metakaryotic stemcells of the precursor lesion. Determination of effective “killing” ofmetakaryotic cells, or the identification of viable and non-viablemetakaryotes, is described herein, and in International ApplicationNumber PCT/US15/20933.

In a particular embodiment of the present invention the mammaliansubject is a human and the disorder characterized by excessivemetakaryotic stem cell growth is cancer, for example, pancreatic cancer.Also encompassed by the present invention are disorders characterized byexcessive metakaryotic stem cell growth such as atheroscleroses,venoscleroses, post-surgical restenoses, scleroderma or diabetesmellitus.

The present invention further encompasses kits comprising dosage formsof one, two or more metakaryocidal agents suitable for treating orpreventing a disorder characterized by excessive metakaryotic stem cellgrowth in a mammalian subject in need comprising two, three, four, fiveor more metakaryocides in the application as a therapy, or when appliedfor disease prevention. Use of a metakaryocidal agent as describedherein, for the preparation of medicament for treating a disordercharacterized by excessive metakaryotic stem cell growth in a mammaliansubject in need thereof by means of a combination metakaryocidal therapyis also encompassed by the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The foregoing will be apparent from the following more particulardescription of example embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingembodiments of the present invention.

FIG. 1 is a photograph illustrating the result of treatment of HT-29cells with zero and increasing levels of metformin for five weeks thustesting metformin as a metakaryocide in vitro.

FIG. 2 is a table illustrating the effects of combinations of metforminconcentrations and durations of treatment of HT-29 cells.

FIG. 3 is a scatterplot illustrating that the simple product of initialconcentration×duration of exposure reveals a two phase survival curvewith ˜10-12% of colony forming stem cells killed by regimens of˜200-2000 μM-weeks.

FIGS. 4A and B show surviving metakaryotic cells in a sample of a lungtumor taken from an adult patient after extended treatment with radio-and chemotherapy. Nuclei of eukaryotic cells are pyknotic but nuclei ofmetakaryotic are not pyknotic, illustrating that present standards ofcare using radio- and chemotherapies kill eukaryotic-non stem cells butnot metakaryotic stem cells in treated tumors.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

The invention is based, at least in part, on Applicants' discovery of agrowing number of drugs already used for diverse medical purposes in theUnited States that have now been found capable of killing growingmetakaryotic stem cells in cell lines derived from adenocarcinomas ofthe human colon (HT-29) and pancreas (CAPAN-1). Most of these drugs weremetakaryocidal at concentrations in cell culture known to be well belowplasma concentrations associated toxic side effects in humans. Some ofthe metakaryocidal drugs identified so far are effective in cell cultureat concentrations in the blood plasma well tolerated by patients usingthese drugs treatment of other maladies, e.g. doxycycline when used asan antibiotic.

From these cell culture experiments, Applicants' previous discovery thathuman stem cells during lung and colon organogenesis haveextraordinarily high mutation rates compared to eukaryotic non-stemcells (Sudo et al., 2008; Kini et al, 2013) and certain mathematicalconsiderations. Applicants have devised a novel course of therapy andprevention comprising a single therapeutically effective drug (e.g., forprevention purposes) or a single, or a set of multidrug treatmentregimens to treat metakaryotic stem cell derived diseases (e.g. diseasesassociated with metakaryotic stem cells). These regimens are designed tokill all, or significantly inhibit the growth of, growing metakaryoticstem cells in mammalian cancers, and their metastases, and tofurthermore capture and kill such residual non-dividing cancer stemcells existing at the initiation of treatment.

The stem cells of human organogenesis and wound healing are noteukaryotic but metakaryotic cells, a cell form previously recognized byApplicants. Applicants subsequently discovered that metakaryotic cellsare also the stem cells of pathological processes includingcarcinogenesis, atherogenesis and post-surgical restenosis.

Metakaryotic cells differ from eukaryotic cells in many characteristicsof cellular and molecular biology but, in particular, are relativelyresistant to killing by agents that kill eukaryotic cells in growthphase such as x-rays, radiomimetic and anti-metabolic agents widely usedto treat cancers. Such eukaryocidal agents kill eukaryotic cells incancerous lesions such as adenocarcinomas in which, for example, theycomprise the cells of the epithelial portion of the lesion. Presentstandards of practice for treatments of tumors and metastases includeregimens of x-irradiation and chemotherapy with one or more drugs thatkill actively dividing eukaryotic cells of the lesion. Treatments ofthis kind kills a large fraction of eukaryotic cells of the lesionrecognized by x-ray or CT scan as shrinkage of the tumor mass in whichmicroscopic examination reveals dead eukaryotic but not metakaryoticcells (FIG. 4). Applicants earlier discovered that the metakaryotic stemcells in cell cultures were not significantly affected by such commonlyapplied cancer treatments. After standard radio- and chemo-therapeutictreatments the metakaryotic stem cells rapidly regenerate the tumor(relapse) leading to death.

Applicants reasoned that agents that killed metakaryotic cells might beused in treating pathological lesions in which metakaryotic cells servedas the stem cells that drove the growth of the lesions. They similarlyreasoned that reduction of the metakaryotic stem cells of apreneoplastic lesion by metakaryocidal treatment could reduce the numberof surviving metakaryotic stem cells so that the subsequent age-specificincidence of the disease could be reduced on the order of a hundred foldusing treatment that would kill >99% survival of metakaryoticprecancerous stem cells within the preneoplastic lesions of a subject'sbody).

Applicants next sought means to test nominated drugs (also referred toherein as candidate agents) using human cell cultures, particularly cellcultures derived from human tumors in which they discovered metakaryoticcells driving net growth and differentiation of such cultures bysymmetric and asymmetric amitotic divisions. Two cell lines inparticular HT-29 (derived from a human colonic adenocarcinoma) andCapan-1 (derived from a human pancreatic adenocarcinoma) were found tohave these characteristics. With these lines they tested nominated drugsby observing (a) evidence of disrupted or dead (pyknotic) metakaryoticcells and (b) reduction of stem cell number evidenced by reduction ofthe fraction of treated cells that gave rise to immortal colonies thatinvariably contained metakaryotic stem cells, as described inInternational Application Number PCT/US15/20933.

Applicants discovered that, unlike the effects of x-rays or drugscommonly used to treat cancers, nominated drugs did not killmetakaryotes in one, two or three days of exposure to concentrationsrepresenting the highest doses known to be tolerated by humans.Unexpectedly, sustained treatment for five consecutive days or longerwas required to observe cytotoxic or immortal colony suppressing effectsof these drugs at concentrations tolerated at plasma levels in humans.Applicants noted that some of the drugs that they found to bemetakaryocidal in their assays had been tested in clinical trials usingshort exposure periods alone or in combination with eukaryocidal“chemotherapeutic” drugs and under the conditions of treatment had beenreported to be ineffective.

Applicants noted that interruption in treatment with metakaryocidaldrugs in cell cultures resulted in greatly reduced metakaryocidaleffect. It was recognized that patient plasma concentrations wouldtherefore need to be maintained at about metakaryocidal concentrationsthroughout the course of treatment in order to be effective in killingall cancer metakaryotic stem cells (also referred to herein as the“course of effective treatment”) or nearly all (>99%) metakaryotic stemcells of precancerous lesions or early pre-atherosclerotic plaques (alsoreferred to herein as course of effective preventive treatment).Monitoring of patients with regard to said metakaryocidal agent plasmaconcentrations would thus be desirable, more probably necessary, asvariation among patients and induction of physiological responsesleading to more rapid drug clearance would be anticipated with prolongedtreatments.

Applicants measured the distribution of mutant colony number and sizesin adult human lungs and discovered the distribution indicated anunexpectedly high and constant mutation rate in the stem cells of humanlung organogenesis, some hundreds to thousands of times higher thanfound by applicants and others in human eukaryotic cells (Sudo et al.,2008. Applicants ascribed these high mutation rates to the peculiar modeof genome replication they had discovered in metakaryotic cells (Thillyet al., 2014).

Applicants had previously studied mutations resulting in loss of drugbinding capacity and drug sensitivity in human cells and found that therate of such mutations was some one hundred times lower than the ratesof mutations that inactivated a gene product (Leong et al., 1985).

Applicants then used their knowledge of mutation rates in humanorganogenesis to hypothesize that a tumor or tumor metastasis wouldcontain many metakaryotic stem cells resistant to any one metakaryocidaldrug that acted by, e.g., binding to and inactivating a target moleculein a metakaryotic cancer stem cell. They thus recognized the possibilitythat multiple metakaryocidal drugs acting, preferably independently,would probably be necessary to kill an effective number (e.g., all)metakaryotic cancer stem cells in a patient or >99% in a preneoplasticlesion or other asymptomatic precursor of a disease in whichmetakaryotic cells serve as the stem cells. Applicants considered thenumber of expected metakaryotic stem cells and the rates of mutations inhuman organogenic metakaryotic stem cells and determined that acombination of drugs, preferably, two, three or four metakaryocidaldrugs would be effective to treat the majority of cancer patients, whilefive or more drugs might be required in a minority of patients.

Applicants had previously considered the period of time between stemcell divisions in adult humans. Based on quantitative enumeration ofdividing and dying eukaryotic cells in the normal colonic epithelium,colonic adenomas and early adenocarcinomas they estimated that theaverage time interval between stem cell divisions (symmetric plusasymmetric divisions) was ˜128, ˜40 and ˜12 days respectively andindirectly estimated it might be on the order of 5-7 days in peritonealmetastases. Because Applicants were aware from cell culture studies thatnot all cells capable of continued growth are actively growing at anytime they hypothesized that treatment with metakaryocidal drugs shouldcontinue a period greater than represented by the estimated average ofabout 12 days between metakaryotic divisions in early colorectal tumors.

Applicants have devised means to use cell populations derived frompathologic lesions comprising these diseases in quantitative assays thatpermit recognition of drugs that in sufficient concentration andduration of uninterrupted application are lethal to metakaryotic stemcells and their immediate precursors. (International Application NumberPCT/US15/20933) They have discovered multiple drugs having thesequalities and found that some of them are drugs that have been used intreating other human disorders in millions of people for many decadesbut that have not been applied so as to provide an uninterrupted periodof application at an effective concentration that together are requiredfor metakaryocidal activity and treatment of metakaryotic diseases.

Applicants' findings and hypotheses indicated that a series oftreatments, e.g., five treatments, with metakaryocidal drugs, with eachdrug treatment maintained in the patient's blood (plasma or serum) at aneffective metakaryocidal concentration for an uninterrupted duration of,for example, about 36 days would en toto expose the metakaryotic cancerstem cells of a patient to about 180 days of effective treatment. Such aregimen of treatment would reasonably be expected to kill all residentmetakaryotic cancer stem cells and as a result offer the possibility ofcomplete elimination of the subject's cancer. Applicants note that thefirst of four of five metakaryocidal treatments is expected to killbetween 99 and 99.99% of all cycling metakaryotic stem cells of thelesion and that the second, third, fourth and any additionaluninterrupted treatments with other metakaryocidal agents are used toachieve the necessary goal of killing all cycling metakaryotic stemcellsof the lesion under treatment. Applicants note that the extended periodrequired for multiple sequential treatments provides a necessaryextended opportunity to kill metakaryotic stem cells of the lesion asmany may have been quiescent at the beginning of metakaryocidaltreatments. Applicants note that treatment of a lesion withmetakaryocides and/or eukaryocides is expected to provoke a woundhealing response among the metakaryotic stem cells of the lesionspecifically provoking quiescent metakaryotic stem cells in the lesionto begin active cell divisions that render them sensitive tometakaryocidal drugs.

Although the period of a course of effective treatment would be ofnecessity continuous and uninterrupted for each metakaryocidal drugadministered, there may be an interval of respite (e.g., a restinginterval) between courses of effective treatment with each of themetakaryocidal drugs. The resting interval may be for a few days, to oneto two weeks, or more up to about one to two months. For example, Drug Acould be administered to the patient for a period of time and dosagesufficient to kill metakaryotes (e.g., for about 36 days) and prior tobeginning treatment with Drug B, a period of one to two weeks couldensue without metakaryocidal treatment.

It is important to note that the treatments described herein alsoencompass treatment with a metakaryocidal agent to destroy most, but notall, of the metakaryotic stem cells of a slowly growing pre-lethallesion such as a colonic adenomatous polyp. Such treatment can beaccomplished by continuous treatment at an effective concentration for aduration of as long as one year to permit action of the drug throughsuccessive intervals of metakaryotic stem cell divisions, symmetric andasymmetric. For example, nine successive intervals between theasymmetric divisions of metakaryotic stem cells of such slowly growinglesions as human colonic adenomas is roughly equivalent to 360 days orone year. Such treatment is expected to greatly reduce the subsequentappearance of tumors throughout the lifetime of the individualundergoing such preventive continuous exposure to a metakaryocide at aneffective plasma concentration.

Disorders to be Treated

As described above, the disorders to be treated encompass any disordercharacterized, or associated with, or derived from, excessivemetakaryotic cell growth. Specifically encompassed by the presentinvention is cancer. A disorder/disease “characterized by excessivemetakaryotic stem cell growth” includes both monoclonal (e.g., cancer,atherosclerosis, venosclerosis) and polyclonal (e.g., restenosis orscleroderma) disorders where metakaryotic cells undergo excessive growthor division-including either asymmetrical or symmetrical divisions. Inparticular embodiments, a monoclonal disorder characterized by excessivemetakaryotic stem cell growth is cancer, including carcinomas, sarcomas,hematological cancers (lymphoma and leukemia), germ cell tumors, andblastomas. In more particular embodiments the cancer is selected frombladder, brain, breast, colon, rectal, endometrial, leukemia, lung,hepatic (e.g., HCC), kidney, melanoma, non-Hodgkin lymphoma, prostate,pancreatic, stomach, and thyroid cancer and combinations thereof. Thecancer may be either localized (non-metastatic) or metastatic.Additional states that may be related to cancer and that can be treatedby the methods provided by the invention include precancerous lesionsand neoplasias. Additional disorders characterized by excessivemetakaryotic stem cell growth include type II diabetes (which Applicantsperceive as a metakaryotic disease based on the identity of itsage-specific mortality to the function observed for many common cancerssuch as colorectal, pancreatic, esophageal and prostate cancers and thefinding by others that at least one form of diabetes is caused by a verysmall, specific form of pituitary neoplastic lesion) endometriosis,polycystic diseases, and benign malignancies.

In still other embodiments, the disorder characterized by excessivemetakaryotic stem cell growth is polyclonal, including disorders such asrestenosis, also known as “galloping atherosclerosis” or the ultimatelylethal disease scleroderma. In the case of vascular restenosesapplicants have specifically observed the creation of smooth musclecells that in overabundance characterize this type of lesion arising byasymmetric amitoses from metakaryotic cells. In the case of scleroderma,applicants have specifically observed metakaryotic stem cells undergoingamitoses and creating the many non-stem cells of these lesions byasymmetric amitoses.

Metakaryotes

“Metakaryote, “metakaryotic stem cell,” and the like refer to cellscharacterized by, inter alia, a bell-shaped nucleus, where the celldivides by amitosis-either symmetrical or asymmetrical. Metakaryoteshave been observed in both animal (insect, mammalian as well as human),and plant cells. Metakaryotes also exhibit a double-stranded hybridRNA/DNA intermediate genome during division. See, e.g., InternationalApplication Publication No. WO 2012/167011, incorporated by reference inits entirety. Metakaryotes can be in the form of either an individualcell with a single nucleus or multinucleate syncytial structures.Metakaryotes are first observed early in human organogenesis in weeks4-7 arising from pre-metakaryotic cells that do not have bell shapednuclei but which develop them before giving rise to two bell shapednuclei by a specialized form of “kissing bell” amitoses (Gostjeva et al.2009). Herein reference to killing of metakaryotic cells is meant toinclude such pre-metakaryotic cells as reasonably expected to occurthat, if surviving treatment can give rise to new metakaryotes andsupport regrowth of the lesion or lesions requiring treatment. Cellculture studies demonstrate that both metakaryotic cells andhypothetical pre-metakaryotic cells, are effectively killed by themetakaryocidal drug treatment as shown for metformin in FIG. 3. Thebasis for this interpretation is that after treatment with the variousmetakaryocides cited, replating of treated cultures discovers no cellscapable of further division as would be expected of survivingmetakaryotic stem cells or hypothetical precursor cells.

The skilled artisan will be able to readily identify metakaryotic stemcells when practicing the methods provided by the invention. Forexample, the methods of identification, screening, diagnosis, prognosisand treatment provided herein can comprise the step of detectingmetakaryotic stem cells from a tissue sample or in cultured cells bydetecting an intermediate dsRNA/DNA duplex genome. Cultured cells orcells from within a tissue samples being visualized by the methods ofthe invention are prepared in a way that substantially preserves theintegrity of nuclear structures in nuclei having maximum diameters up toabout 10, 20, 30, 40, 50, 60, or 70 microns—and in more particularembodiments up to about 50 microns. Methods for preparing cells are alsodescribed in U.S. Pat. No. 7,427,502, the teachings of which areincorporated by reference in their entirety. In certain embodiments, thepreparation substantially preserves the integrity of nuclear structuresin metakaryotic nuclei of about 10-15 microns. For example, in someembodiments a tissue sample may be analyzed as a preparation of at leastabout 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400,450, 500, 750, 1000, 1250, 1500 or more microns in thickness. In certainembodiments, a tissue sample is macerated by, for example, incubation inabout 45% (e.g., about 25, 30, 35, 40, 42, 45, 47, 50, 55, 60 or 65%)acetic acid in preparation for analysis. Alternately, the sample may bemacerated by collagenase or other suitable protease (Gostjeva et al.,2009).

In some embodiments, to further facilitate detection of metakaryotes,cultured cells or tissue samples can be stained. In particularembodiments, the staining can comprise staining with, for example, aSchiffs base reagent, Feulgen reagent, or fuchsin. In more particularembodiments, the tissue sample may be further stained with a secondstain. In still more particular embodiments, the second stain may beGiemsa stain.

In certain embodiments, metakaryotic stem cells can be detected by thefluorescence of their cytoplasm, following treatment with anon-fluorescent stain, such as Schiffs reagent. See, e.g., U.S. PatentApplication Publication No. 2010/0075366 A1, including Example 5, FIGS.20-27, and their descriptions, all of which are incorporated byreference. Metakaryotes involved in wound healing disorders, whichgenerally do not exhibit Feulgen reagent-induced fluorescence in theirballoon-shaped cytoplasmic structures, are described in InternationalApplication Publication No. WO 2012/061073, incorporated by reference inits entirety.

Treatment Modalities

A “combination metakaryocidal therapy” is a regimen of two or more (e.g.2, 3, 4, 5, 6, or more) “metakaryocidal treatments”—i.e. chemical agents(such as a small molecule or biologic; “metakaryocidal agents”) orenvironmental conditions (i.e. changes in temperature, pH, pressure,pO2, and combinations thereof; “metakaryocidal conditions”) or acombination of chemical agent(s) and environmental condition(s)—thatinhibit the growth or proliferation of cycling (i.e., non-dormant)metakaryotes and encompasses agents that reduce the number ofmetakaryotes (e.g., by killing the metakaryotes) or blocking an increasein the number of these cells—i.e., agents or treatments termed“metakaryostatic.” Treatments can also include irradiation such asx-rays. Metakaryocidal treatments preferentially inhibit the growth orproliferation of cycling metakaryotes over cycling eukaryotes (e.g., by50, 60, 70, 80, 90, 95%, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000-fold,or more). In certain embodiments, the regimen comprises concurrentmetakaryocidal treatments. In other embodiments, the regimen comprisessequential metakaryocidal treatments. In still other embodiments, theregimen comprises both concurrent and sequential metakaryocidaltreatments—e.g., a regimen comprising three metakaryocidal treatments,A, B, and C, may, in certain embodiments, entail concurrent treatmentwith two agents (e.g., A and B, A and C, or B and C) preceded orfollowed by treatment with the third agent (i.e., C, B, or A in theforegoing examples).

An “uninterrupted therapeutically effective combination metakaryocidaltherapy” is a combination metakaryocidal therapy where each and all ofthe metakaryocidal drug treatments of the combination are maintainedwithin an “effective range of metakaryocidal activity”—i.e., at or abovethe minimally effective (i.e., metakaryocidal) dose of a metakaryocidalagent and at or below the maximum tolerable dose of the metakaryocidalagent-over the entire course of effective therapy period; e.g., in someembodiments, for the entire approximately −7 week period for eachmetakaryocidal treatment and over the entire combination of two or more(e.g., 4-6) treatments in the combination metakaryocidal therapy. Thisteaching specifically addresses the finding that under exposure to aparticular metakaryocidal drug interruption of treatment greatlydiminishes or even eliminates the effectiveness of treatment with thatdrug. However, a resting period between separate treatments withseparate metakaryocidal drugs may in some cases be medically desirable,e.g., wherein surgery becomes necessary or when extended periods ofcombined metakaryocidal treatment induce presently unexpected sideeffects in normal tissues. Limits on such periods between metakaryocidaltreatments are bounded by the expectation that overly extended periodswould permit regrowth of metakaryotic cells and their precursors notkilled by prior treatment.

Metakaryocidal agents include certain NSAIDs (non-steroidalanti-inflammatory agents) and other analgesics, including agents thatinhibit both Cox-1 and Cox2 (such as acetaminophen or aspirin), or Cox-2specific inhibitors (e.g., celecoxib); metformins; tetracyclines (e.g.,doxycycline); ACE (angiotensin-converting enzyme) inhibitors (such ascaptopril); prostacyclin (PGI2) analogs (such as treprostinil, alsoknown as REMODULIN®); sulfonylureas, phenylalkylamines (such asverapamil) and salinomycin.

A “subject” (i.e., to be treated by the methods provided by theinvention) refers to a mammal, including primates (e.g., humans ormonkeys), cows, sheep, goats, horses, dogs, cats, rabbits, guinea pigs,rats, mice or other bovine, ovine, equine, canine, feline, rodent ormurine species. Examples of suitable subjects include, but are notlimited to, human patients. In particular embodiments, the subject to betreated by the methods provided by the invention is human and can bemale or female and may be used in treatments of adult humans (earlyadult, middle-age, or geriatric, e.g., at least or about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 18, 20, 21, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 105 years old, or more) and other animals.However, as metakaryotes are also the stem cells of normal growth anddevelopment and wound healing of humans and other animals (Gostjeva etal., 2009) use in humans or other animals that have not reached maturitycannot be recommended: e.g., prenatal, neonatal, infant, toddler,juvenile patients. In like fashion, treatment of pregnant women withmetakaryocides is expected to endanger the fetus for any metakaryocidalagent that reaches the fetus. An apparent important exception is themetakaryocidal drug metformin that does not cross the placental barrierand has been widely used to treat polycystic disease in pregnant women.Similarly, metakaryotic drugs should not be used for a periodimmediately before and after surgery, a caveat generally recognized inpatients using drugs such as metformin now recognized by Applicants asmetakaryocidal but without the understanding that the drug kills orretards the growth of metakaryotic stem cells essential for woundhealing. Similarly, it is apprehended that in the use of sequentialtreatments with combinations of metakaryocides certain unknown roles ofmetakaryotes in maintaining adult tissue structures may become apparentsuch that as in the case of pre-surgical care, a period of time, e.g.,two weeks between treatments with separate metakaryotic drugs may bepreferred.

As used herein, the terms “treat,” “treating,” or “treatment,” mean tocounteract or prevent a medical condition (e.g., cancer,atherosclerosis, restenosis) to the extent that the medical condition isimproved according to a clinically-acceptable standard.

The terms “prevent,” “preventing,” or “prevention,” as used herein, meanreducing the probability/likelihood, progression, onset, risk orseverity of a disorder-including, for example, cancer, atherosclerosis,or restenosis.

As used herein, a “therapeutically effective amount” is an amountsufficient to achieve the desired therapeutic or prophylactic effectunder the conditions of administration, such as an amount sufficient toinhibit (e.g., reduce, prevent), e.g., a condition characterized byexcessive metakaryotic stem cell growth. The effectiveness of a therapycan be determined by one skilled in the art using standard measures androutine methods.

In certain embodiments of the methods provided by the invention, thecombination metakaryocidal therapy comprises administering one or moremetakaryocidal agents so as to achieve plasma concentrations, in thesubject being treated, of the agents “substantially similar” (i.e.,within about 4-fold, 2-fold, or 1-fold but not lower than the effectivein vitro metakaryocidal concentrations of the agents in cells inculture, such as in HT-29 or CAPAN-1 cells. “Metakaryocidalconcentrations” of an agent are those that achieve at least a 99, 99.9,99.99%, or more, reduction in the number of metakaryotes, preferentiallyover eukaryotes, in an HT-29 or CAPAN-1 culture (e.g., in some casesresulting in about 10-4 survival of metakaryotes), within about 10interdivision periods, e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 weeks of “substantially constant” patient plasma levels—i.e.,maintaining a concentration of the agent in the effective concentrationrange in patient's plasma. In more particular embodiments, themetakaryocidal dose in HT-29 or CAPAN-1 cells is determined by an assayas illustrated by the Exemplification.

In certain embodiments, the metakaryocidal therapy comprisesadministering an effective amount of one or more of captopril,celecoxib, doxycycline, metformin, verapamil, or acetaminophen, i.e., atleast 1, 2, 3, 4, 5, or all 6 of these agents. Additional agents arebeing discovered by applicants through continued trials of differentagents such that many more drugs are expected to be found to bemetakaryocidal in concentration ranges well tolerated as concentrationsin plasma. In more particular embodiments, the effective amount ofcaptopril achieves a plasma concentration of about 0.25 μM (about, e.g.,0.025, 0.06, 0.09, 0.125, 0.19, 0.25, 0.31, 0.37, 0.43, 0.50, 0.56,0.62, 0.68, 0.75, 1.0 or 2.5 μM; e.g., about 0.125-0.50 μM); theeffective amount of celecoxib achieves a plasma concentration of about25.0 μM (about, e.g., 2.5, 6.0, 9.0, 12.5, 19.0, 25.0, 31.0, 37.0, 43.0,50.0, 56.0, 62.0, 68.0, 75.0, or 250 μM; e.g., about 12.5-50.0 μM), theeffective amount of doxycycline achieves a plasma concentration of about5.0 μM (about, e.g., 0.5, 1.25, 1.85, 2.5, 3.75, 5.0, 6.25, 7.5, 8.75,10.0, 15.0, 20.0, or 50 μM; e.g., about 2.5-10.0 μM) the effectiveamount of metformin achieves a plasma concentration of about 400.0 μM(about, e.g., 40, 100, 150, 200, 300, 400, 500, 600, 700, 800, 1200,1600, 4000 μM; e.g., about 200-800 μM), and the effective amount ofverapamil achieves a plasma concentration of about 4.0 μM (about, e.g.,0.4, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0, 16.0, 40.0 μM; e.g., 2.0-8.0μM). In still more particular embodiments 4-6 (e.g., 5) of these agentsare administered sequentially (e.g., one-at-a-time) for about 4-6 (e.g.,5) weeks each over a total period of about 16-36 weeks (for example,about 25 weeks). By monitoring plasma levels of the metakaryocidalagents (or one or more metabolites thereof), a caregiver can maintain atherapeutically effective dosage of the agent, accounting for thesubject's metabolism by adjusting the administered dose of the agent. Incertain embodiments, the plasma concentration of the metakaryocidalagent is kept substantially constant, as defined above. The skilledartisan will appreciate that the target plasma concentrations ofmetakaryocidal agents and the target exposure period may be adjusted soas to achieve a similar time-dosage, e.g., by maintaining a lower plasmaconcentration of the agent for a longer period of time or, conversely, ahigher plasma concentration for a shorter period of time. In particularembodiments, the target plasma concentration is less than −25% of themaximum tolerated level for humans or other treated animals. Applicantsrecognize that distribution and therefore the effectiveness ofmetakaryocides to the metakaryotic stem cells to the lesions targetedfor treatment of a subject is a function of processes of internaldistribution, metabolism and clearance in the body and treated lesionsthat are not yet understood. The future study of pharmacokinetic andpharmacodynamic parameters that govern the levels of metakaryocidaldrugs to their target metakaryotic stem cells are expected to add usefulunderstanding regarding the use of metakaryocidal agent therapies intreatment and prevention of diseases in which metakaryotic stem cellsare the driving force of lesion growth and differentiation.

The therapeutically effective plasma concentrations of themetakaryocidal agents are maintained via accepted means, such asperiodic oral, intravenous, mucosal (e.g., rectally, nasally, or byinhalation) or transdermal (including IP) administration. For example,in certain embodiments, the metakaryocidal agents are administeredorally and, in more particular embodiments, are administered orallyapproximately every, e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, or24 hours, or about 1, 2, 3, 4, 5, 6, 7 days, depending on, inter alia,the serum half-life for the subject and available dosage forms of theagent. In other embodiments, the agent is administered by a pump(optionally including a sensor to monitor plasma levels of the agent, ametabolite of the agent, or another correlate for the concentration ofthe agent) or a delayed release modality. In a preferred embodimentcontinuous infusion of metakaryocidal drugs are used with portableinfusion pumps equipped with means to recognize, promptly respond to andrecord an infusion failure so as to ensure uninterrupted drug delivery.

In the methods provided by the invention, the metakaryocidal treatmentsof the combination metakaryocidal therapy are each administered for aperiod corresponding to about 2, 3, 4, 5, 6, 7, 8, 9, or 10 metakaryotedoubling times (e.g., about 4-6 doubling times) for the particular tumorin the subject, e.g. about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13weeks, e.g., about 3-7 weeks, e.g., about 5 weeks. In some embodiments,the treatments are each administered for this period and, as previouslynoted, the treatments can for some drug combinations be administeredconcurrently or for all series of drug treatments sequentially.

Effective dosages of metakaryocidal agents can also be furtherapproximated by using effective dosages achieved in one animal andconverting the dose for use in another animal, including humans, usingconversion factors known in the art. See. e.g., Reagan-Shaw et al.,FASEB J. 22:659-61 (2008); Schein et al., Clin. Pharmacol. Ther. 11:3-40 (1970); and Freireich et al., Cancer Chemother. Reports 50(4):219244 (1966). For example, human equivalent dosing (HED) in mg/kg based onanimal dosing may be given by the following equation: HED (mg/kg)=animaldose (mg/kg)×(K^(manimal)/K^(mhuman)), where Km=weight/surface area(kg/^(m2)). Exemplary conversion factors based on the above equation areshown in the following table.

From: Mouse Rat Monkey Dog Human To: (20 g) (150 g) (3.5 kg) (8 kg) (60kg) Mouse 1 0.5 0.25 0.17 0.08 Rat 2 1 0.5 0.25 0.14 Monkey 4 2 1 0.60.33 Dog 6 4 1.7 1 0.5 Human 12 7 3 2 1

In certain embodiments, the subject being treated by the methodsprovided by the invention, e.g., where the condition characterized byexcessive metakaryotic stem cell growth is cancer, is also beingtreated, either concurrently or sequentially with one or moreeukaryocidal treatments. “Eukaryocidal treatment” means conventionalcancer treatments that kill or inhibit the growth of non-metakaryoticcancer cells. Exemplary eukaryocidal treatments include chemotherapy,radiation therapy, surgery, et cetera, such as treatment withgemcitabine (see, e.g., PubChem 60750, 60749, 11599950, 6420157,44558863, 9828310), 5-fluorouracil (“5-FU”, and related compounds; see,e.g., PubChem 3385, 25244711, 8642), methotrexate (see, e.g., PubChem126941, 4112, 72440, 165528, 10713), a cis-platinum, or certainmonoclonal antibodies In more particular embodiments, the treatmentcomprises surgery, such as a Whipple procedure and/or hepatic arteryembolization for a subject with pancreatic cancer. In other embodiments,the eukaryocidal treatment comprises a gemcitabine treatment, such as agemcitabine combination therapy (e.g., in combination with one or moreof erlotinib (see, e.g., PubChem 176870, 176871, 18924996, 11599950);oxaliplatin (see, e.g., PubChem 5310940, 77994, 9887054); or 5-FU).

Agents (either metakaryocidal or eukaryocidal) for use in the methodsprovided by the invention can be delivered by any suitable route and inany suitable form. The pharmaceutical preparations disclosed herein areprepared in accordance with standard procedures and are administered atdosages that are selected to reduce, prevent, or eliminate, or to slowor halt the progression of, the condition being treated (see, e.g.,Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., and Goodman and Gilman's The Pharmaceutical Basis of Therapeutics,McGraw-Hill, New York, N.Y., the contents of which are incorporatedherein by reference, for a general description of the methods foradministering various agents for human therapy). The compositions of acompound for use in the methods provided by the invention can bedelivered using controlled or sustained-release delivery systems (e.g.,capsules, biodegradable matrices). Exemplary delayed-release deliverysystems for drug delivery that would be suitable for administration ofthe compositions of the disclosed compounds are described in U.S. Pat.No. 5,990,092 (issued to Walsh); U.S. Pat. No. 5,039,660 (issued toLeonard); U.S. Pat. No. 4,452,775 (issued to Kent); and U.S. Pat. No.3,854,480 (issued to Zaffaroni), the entire teachings of which areincorporated herein by reference.

For preparing pharmaceutical compositions for use in the methods andkits provided by the invention, pharmaceutically acceptable carriers caneither be solid or liquid. Solid form preparations include powders,tablets, pills, capsules, cachets, suppositories, and dispersiblegranules. For example, the compounds of the present invention may be inpowder form for reconstitution at the time of delivery. A solid carriercan be one or more substances which may also act as diluents, flavoringagents, solubilizers, lubricants, suspending agents, binders,preservatives, tablet disintegrating agents, or an encapsulatingmaterial. In powders, the carrier is a finely divided solid which is ina mixture with the finely divided active ingredient.

In tablets, the active ingredient is mixed with the carrier having thenecessary binding properties in suitable proportions and compacted inthe shape and size desired.

The powders and tablets preferably contain from about one to aboutseventy percent of the active ingredient. Suitable carriers aremagnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin,dextrin, starch, gelatin, tragacanth, methylcellulose, sodiumcaboxymethycellulose, a low-melting wax, cocoa butter, and the like.Tablets, powders, cachets, lozenges, fast-melt strips, capsules andpills can be used as solid dosage forms containing the active ingredientsuitable for oral administration.

Liquid form preparations include solutions, suspensions, retentionenemas, and emulsions, for example, water or water propylene glycolsolutions. For parenteral injection, liquid preparations can beformulated in solution in aqueous polyethylene glycol solution.

Aqueous solutions suitable for oral administration can be prepared bydissolving the active ingredient in water and adding suitable colorants,flavors, stabilizing agents, and thickening agents as desired. Aqueoussuspensions for oral administration can be prepared by dispersing thefinely divided active ingredient in water with viscous material, such asnatural or synthetic gums, resins, methylcellulose, sodiumcarboxymethylcellulose, and other well-known suspending agents.

The pharmaceutical compositions for use in the methods and kits providedby the invention are preferably in unit dosage form. In such form, thecomposition is subdivided into unit doses containing appropriatequantities of the active ingredient. The unit dosage form can be apackaged preparation, the package containing discrete quantities of, forexample, tablets, powders, and capsules in vials or ampules. Also, theunit dosage form can be a tablet, cachet, capsule, or lozenge itself, orit can be the appropriate amount of any of these in packaged form. Thequantity of active ingredient in a unit dose preparation may be variedor adjusted to achieve the target plasma concentrations describe, above,e.g., from about 0.1 mg to about 1000.0 mg, e.g., from about 0.1 mg toabout 100 mg or from about 1.0 mg to about 1000 mg. The dosages,however, may be varied depending upon the requirements of the patient,the severity of the condition being treated, the compound and the routeof administration being employed. Determination of the proper dosage fora particular situation is within the skill in the art. Also, thepharmaceutical composition may contain, if desired, other compatibletherapeutic agents.

In general, the methods for delivering the disclosed compounds andpharmaceutical compositions of the invention in vivo utilizeart-recognized protocols for delivering the agent with the onlysubstantial procedural modification being the substitution of thecompounds represented by any one of the disclosed compounds for thedrugs in the art-recognized protocols.

The compounds for use in the methods and kits provided by the inventionmay be administered by any route, preferably in the form of apharmaceutical composition adapted to such a route, and would bedependent on the condition being treated. The compounds and compositionsmay, for example, be administered intravascularly, intramuscularly,subcutaneously, intraperitoneally, orally or topically. It will beobvious to those skilled in the art that the following dosage forms maycomprise as the active ingredient, either compounds or a correspondingpharmaceutically acceptable salt of a compound of the present invention.A preferred method of administration for the compounds of the inventionis oral administration.

In some embodiments, the composition may be administered parenterallyvia injection. Parenteral administration can include, for example,intraarticular, intramuscular, intravenous, intraventricular,intraarterial, intrathecal, subcutaneous, or intraperitonealadministration. Formulations for parenteral administration may be in theform of aqueous or non-aqueous isotonic sterile injection solutions orsuspensions. These solutions or suspensions may be prepared from sterilepowders or granules having one or more of the carriers mentioned for usein the formulations for oral administration. The compounds may bedissolved in polyethylene glycol, propylene glycol, ethanol, corn oil,benzyl alcohol, sodium chloride, and/or various buffers (e.g., sodiumbicarbonate, sodium hydroxide).

For oral administration, the pharmaceutical compositions may be in theform of, for example, a tablet, capsule, suspension or liquid. Thecomposition is preferably made in the form of a dosage unit containing atherapeutically effective amount of the active ingredient. Examples ofsuch dosage units are tablets and capsules. For therapeutic purposes,the tablets and capsules can contain, in addition to the activeingredient, conventional carriers such as binding agents, for example,acacia gum, gelatin, polyvinylpyrrolidone, sorbitol, or tragacanth;fillers, for example, calcium phosphate, glycine, lactose, maize-starch,sorbitol, or sucrose; lubricants, for example, magnesium stearate,polyethylene glycol, silica, or talc; disintegrants, for example potatostarch, flavoring or coloring agents, or acceptable wetting agents. Oralliquid preparations generally in the form of aqueous or oily solutions,suspensions, emulsions, syrups or elixirs may contain conventionaladditives such as suspending agents, emulsifying agents, non-aqueousagents, preservatives, coloring agents and flavoring agents. Examples ofadditives for liquid preparations include acacia, almond oil, ethylalcohol, fractionated coconut oil, gelatin, glucose syrup, glycerin,hydrogenated edible fats, lecithin, methyl cellulose, methyl or propylpara-hydroxybenzoate, propylene glycol, sorbitol, or sorbic acid.

For topical use the compounds of the present invention, specificallyanticipated in the case of scleroderma, may also be prepared in suitableforms to be applied to the skin, or mucus membranes of the nose andthroat, and may take the form of creams, ointments, liquid sprays orinhalants, lozenges, or throat paints. Such topical formulations furthercan include chemical compounds such as dimethylsulfoxide (DMSO) tofacilitate surface penetration of the active ingredient. Suitablecarriers for topical administration include oil-in-water or water-in-oilemulsions using mineral oils, petrolatum and the like, as well as gelssuch as hydrogel. Alternative topical formulations include shampoopreparations, oral pastes and mouthwash.

For application to the eyes or ears, the compounds of the presentinvention may be presented in liquid or semi-liquid form formulated inhydrophobic or hydrophilic bases as ointments, creams, lotions, paintsor powders.

For rectal administration the compounds of the present invention may beadministered in the form of suppositories admixed with conventionalcarriers such as cocoa butter, wax or other glyceride. For preparingsuppositories, a low-melting wax, such as a mixture of fatty acidglycerides or cocoa butter, is first-melted and the active ingredient isdispersed homogeneously therein, as by stirring. The molten homogeneousmixture is then poured into convenient sized molds, allowed to cool, andthereby to solidify.

Delivery can also be by use of a timed release or sustained releasematrix delivery systems, or by onsite delivery using micelles, gels andliposomes. Nebulizing devices, powder inhalers, and aerosolizedsolutions are representative of methods that may be used to administersuch preparations to the respiratory tract.

Exemplification

The literature describing cell lines derived from human tumors usuallyindicates that several forms of colonies may be distinguished a singlyseeded cells grow to localized colonies of ˜64 to thousands of cells.HT-29 and CAPAN-1 cell are not exceptions.

The metakaryotic and pre-metakaryotic cells of cell lines derived fromhuman tumors are capable of unlimited division when plated as singlecells on plastic or glass surfaces. However, most cells in such celllines are eukaryotic cells capable of limited cell growth which growthcannot give rise to immortal colonies or populations of cells. Thisquality permits the line to be used in quantitative assays of cellkilling important for distinguishing among agents and regimens that killmetakaryotic cells in preference to eukaryotic cells and vice versa.

Missing from the present discussion in the literature of both tumors andtumor derived lines is that of stem cells, metakaryotic and possiblypre-metakaryotic, increase their own number by symmetrical amitoticdivisions and unknown modes of division, respectively. They are alsoresponsible for the increase in tumor mass by the creation of firsttransition eukaryotic cells that by a series of binomial mitoses createa terminal cell population. When organized in adult tissue such as inthe colonic crypts, the assemblage of maintenance stem cell and firstthrough terminal cells create a turnover unit in which the apparentlyprogrammed cell deaths of terminal cells are matched by divisions oflower tier eukaryotic cells and the single maintenance stem cell at thecrypt base. The turnover unit cell number remains approximately constantand the time interval between divisions of any cell is the same as thelifetime of any terminal cell. In the colon, for instance, Furth's datapermitted calculation that this interval is ˜128 days (Herrero-Jiminezet al., 1998, 2000).

As noted above, microtiter well studies of single cells of theexponentially growing HT-29 cell line indicated that this line closelyimitates the growth of colonic crypts. Single cells betray differentfates. A small fraction (˜5-15% under present conditions) form colonies,button shaped or disperse, that express metakaryotic cell forms and growto >2¹³ cells and grow to millions of cells (>2²⁰) when trypsinized andtransferred in a series of T-flasks. Applicants interpret these coloniesand derived immortal populations to be the descendants of singlemetakaryotic or pre-metakaryotic stem cells.

However, most single cells give rise to colonies that range from 1 to˜2000 (2¹¹) cells that do not contain any visible metakaryotic cells andon trypsinized transfer do not increase in cell number. Applicantsinterpret these colonies that do not divide when placed as single,attached cells in microwells to be elements of turnover units containing˜2¹¹ terminal cells/unit. Applicants interpret the colonies of ˜2¹¹cells that do not grow on transfer to be derived from the second tier ofthe turnover unit which contains two cells. Colonies of limited sizebetween 1 and 2¹¹ cells are interpreted to be the products of growth oftransition cell tiers three through eleven.

In this reproducible behavior the human tumor cell lines offered a meansto test agents for metakaryocidal activity: metakaryocidal agents andregimens would reduce or eliminate the appearance of large colonies thatcould be propagated by serial trypsinized transfer. Metakaryocidalaction could also be recognized by reduction of total colony numberafter trypsinized transfer of the HT-29 and like lines by the reductionin total colonies after treatment from that found in untreated control;flasks to about 90% of that number, the 10% difference representing thecolonies that save for treatment would have arisen from a metakaryoticstem cell. An example of this kind of assay is illustrated in FIGS. 1, 2and 3.

Multiple drugs have been tested based on their reported ability tointerfere with wound healing (phleomycin) suppress cancer rates in thediabetic human population (metformin, sulphonyl ureas) or cure animalcancers in combination with known eukaryocidal drugs which alone are noteffective (metformin, doxycycline). In particular drugs evaluated werereported to overcome “multidrug resistance” giving special priority todrugs that suppressed “multidrug resistance” (MDR). Cytotological andclonal assays have, so far, yielded equivalent results. Drug regimensthat resulted in the appearance of pyknotic bell shaped nuclei alsokilled off cells capable of forming immortal colonies in HT-29 cellcultures. Cytologic results in HT-29 yielded identical results in theCAPAN-1 cell line.

Shown in FIGS. 1-3 are images of quantitative trials of differentregimens (initial concentration, duration of exposure) of metformin inclonal assays using HT-29 cells. The image in FIG. 1 is that of a set ofT-flasks with HT-29 cells surviving to form large colonies some fiveweeks after beginning of treatment with metformin under the conditionsindicated. FIG. 1 is a photograph illustrating the experimental designfor testing metformin as a metakaryocide in vitro. In the concentrationrange 100-400 micromolar an ˜10% reduction in large colonies is observedthat corresponds to immortal colonies in which metakaryotic cells areobserved as stem cells. Large colonies surviving >100 micromolar are notimmortal and do not contain metakaryotic stem cells.

FIG. 2 depicts a table of the recorded colony counts on each of theflasks of the first image. The image in FIG. 2 illustrates the reductionof large colonies forming five weeks after seeding HT-29 cells treatedfrom day one for one, two or five weeks with the concentrations ofmetformin indicated. Total large colony numbers were reduced to a levelabout 10% less than in untreated control flasks at theconcentration×duration of exposure treatments indicated with greencolony counts. Applicants have discovered that ˜10% reduction in largecolonies observed as indicated corresponds to immortal colonies in whichmetakaryotic cells are observed as stem cells. Large colonies survivingtreatment for >1600 micromolar for one week exposure or 800 micromolarfor five weeks exposure are not immortal and do not contain metakaryoticstem cells. The cells in these surviving large colonies indicated by redtype are wholly eukaryotic, grow to a terminal cell stage and do notfurther grow when transferred to new culture surfaces.

The graph in FIG. 3 is a scatter plot representation of the data of FIG.2, showing the regimen dependent survival of what we interpreted to bemetakaryotic stem cells in regimens that kill the metakaryotes plated.The Y-axis is the number of colonies on each test flask enumerated inFIG. 2 while the X-axis is the product of metformin concentration(micromolar) times the duration of the HT-29 cells to the drug (weeks).Typical of all metakaryocidal drugs discovered by applicants using HT-29cells to date, the number of large colonies containing metakaryoticcells and capable of immortal growth in untreated flasks comprising some10% of all large colonies observed (FIG. 1) decreases as a function ofdrug concentration×duration of exposure until no large colonies capableof immortal growth survive. For this drug this elimination ofmetakaryotic cell-containing immortal colonies is reached by ˜200micromolar-weeks while killing of mortal colonies is not observed untilabout 2000 micromolar weeks. Here it appears that a regimen holdingpatient plasma levels at or somewhat above 200 micromolar for anuninterrupted five week period would kill the cycling metakaryotic cellsin a pathologic lesion in which metakaryotes comprise a stem celllineage save for metakaryotic stem cells resistant to the drug metforminas are expected in tumors and metastases because of the high rate ofmutation discovered in human organogenic stem cells (Sudo et al., 2008).

From the data of FIG. 3 a scatterplot illustrating that the log survivalversus concentration×duration of metformin exposure may be constructedand used to calculate the treatment regimen for treating metakaryoticdiseases. In this example the metformin-sensitive population(metakaryotic cells) has a survival fraction of 10⁻² at ˜200 μM weeks.From this, it is expected that a treatment of ˜400 μM weeks couldachieve a 10⁻⁴ survival, limited by hypothetical metformin-resistantmutants expected to arise at >10⁻⁴ in metakaryotic disease lesions suchas tumors or atherosclerotic plaques.

In tests of some sixteen drugs the choice of which was guided byinferences of applicants suggesting they might be metakaryocidal eighthave demonstrated survival vs. concentration×duration function similarto that observed with metformin, e.g., doxicycline, verapamil andsalinomycin, while one, reserpine, proved to have no detectable effectas a metakaryocide.

It should be understood that for all numerical bounds describing someparameter in this application, such as “about,” “at least,” “less than,”and “more than,” the description also necessarily encompasses any rangebounded by the recited values. Accordingly, for example, the descriptionat least 1, 2, 3, 4, or 5 also describes, inter alia, the ranges 1-2,1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5, et cetera.

For all patents, applications, or other reference cited herein, such asnon-patent literature and reference sequence information, it should beunderstood that it is incorporated by reference in its entirety for allpurposes as well as for the proposition that is recited. Where anyconflict exits between a document incorporated by reference and thepresent application, this application will control. All informationassociated with reference gene sequences disclosed in this application,such as GeneIDs, Unigene IDs, or HomoloGene ID, or accession numbers(typically referencing NCBI accession numbers), including, for example,genomic loci, genomic sequences, functional annotations, allelicvariants, and reference mRNA (including, e.g., exon boundaries orresponse elements) and protein sequences (such as conserved domainstructures), as well as chemical references (e.g., Pub Chem compound,Pub Chem substance, or Pub Chem Bioassay entries, including theannotations therein, such as structures and assays, etcetera) are herebyincorporated by reference in their entirety.

Headings used in this application are for convenience only and do notaffect the interpretation of this application.

Preferred features of each of the aspects provided by the invention areapplicable to all of the other aspects of the invention mutatis mutandisand, without limitation, are exemplified by the dependent claims andalso encompass combinations and permutations of individual features(e.g., elements, including numerical ranges and exemplary embodiments)of particular embodiments and aspects of the invention including theworking examples. For example, particular experimental parametersexemplified in the working examples can be adapted for use in theclaimed invention piecemeal without departing from the invention. Forexample, for materials that are disclosed, while specific reference ofeach various individual and collective combinations and permutation ofthese compounds may not be explicitly disclosed, each is specificallycontemplated and described herein. Thus, if a class of elements A, B,and C are disclosed as well as a class of elements D, E, and F and anexample of a combination of elements, A-D is disclosed, then even ifeach is not individually recited, each is individually and collectivelycontemplated. Thus, is this example, each of the combinations A-E, A-F,B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated andshould be considered disclosed from disclosure of A, B, and C: D, E, andF: and the example combination A-D. Likewise, any subset or combinationof these is also specifically contemplated and disclosed. Thus, forexample, the sub-group of A-E, B-F, and C-E are specificallycontemplated and should be considered disclosed from disclosure of A, B,and C; D, E, and F; and the example combination A-D. This conceptapplies to all aspects of this application including, elements of acomposition of matter and steps of method of making or using thecompositions.

The forgoing aspects of the invention, as recognized by the personhaving ordinary skill in the art following the teachings of thespecification, can be claimed in any combination or permutation to theextent that they are novel and non-obvious over the prior art-thus tothe extent an element is described in one or more references known tothe person having ordinary skill in the art, they may be excluded fromthe claimed invention by, inter alia, a negative proviso or disclaimerof the feature or combination of features.

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While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A method of treating a disorder characterized byexcessive metakaryotic stem cell growth in a mammalian subject in needthereof comprising providing a therapeutically effective combinationmetakaryocidal therapy, in which exposure of the subject to eachseparate metakaryocidal drug is uninterrupted, thereby treating thedisorder.
 2. The method of claim 1, wherein the subject receivesconcurrent or sequential eukaryocidal treatment.
 3. A method of treatinga disorder characterized by excessive metakaryotic stem cell growth in amammalian subject in need thereof comprising sequential eukaryocidaltreatment.
 4. The method of claim 1, wherein the combinationmetakaryocidal therapy comprises concurrent or sequential administrationof three or more metakaryocidal treatments.
 5. The method of claim 4,wherein the combination metakaryocidal therapy comprises concurrent orsequential administration of four or more metakaryocidal treatments. 6.The method of claim 5 wherein the combination metakaryocidal therapycomprises concurrent or sequential administration of five or moremetakaryocidal treatments.
 7. The method of claim 6, wherein thecombination metakaryocidal therapy comprises concurrent or sequentialadministration of six or more metakaryocidal treatments.
 8. The methodof claim 1, wherein the combination metakaryocidal therapy comprisesadministering two or more metakaryocidal agents so as to achieve plasmaconcentrations of the agents for an uninterrupted duration substantiallysimilar to the in vitro metakaryocidal concentrations and uninterrupteddurations of the agents found to be metakaryocidal in HT-29 or CAPAN-1cells.
 9. The method of claim 1, wherein the combination metakaryocidaltherapy comprises administering an effective amount of three or more ofcaptopril, celecoxib, doxycycline, metformin, verapamil, oracetaminophen.
 10. The method of claim 1, wherein the combinationmetakaryocidal therapy comprises administering an effective amount offour or more of captopril, celecoxib, doxycycline, metformin, orverapamil.
 11. The method of claim 1, wherein the combinationmetakaryocidal therapy comprises administering an effective amount ofcaptopril, celecoxib, doxycycline, metformin, and verapamil.
 12. Themethod of claim 11, where the effective amount of captopril achieves aplasma concentration of about 0.125 to about 0.5 μM (e.g., about 0.25μM), the effective amount of celecoxib achieves a plasma concentrationof about 12.5 to about 50 μM (e.g., about 25 μM), the effective amountof doxycycline achieves a plasma concentration of about 2.5 to about 10μM (e.g., about 5 μM), the effective amount of metformin achieves aplasma concentration of about 200 to about 800 μM (e.g., about 400 μM),and the effective amount of verapamil achieves a plasma concentration ofabout 2 to about 8 μM (e.g., about 4.0 μM).
 13. The method of claim 1,wherein the metakaryocidal treatments of the combination metakaryocidaltherapy are each administered for a period of about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, or 13 weeks.
 14. The method of claim 13, wherein themetakaryocidal treatments of the combination metakaryocidal therapy areeach administered for a period of about 3-7 weeks.
 15. The method ofclaim 1, wherein the metakaryocidal treatments of the combinationmetakaryocidal therapy are administered sequentially.
 16. The method ofclaim 1, wherein the disorder characterized by excessive metakaryoticstem cell growth is cancer.
 17. The method of claim 16, wherein thecancer is pancreatic cancer.
 18. The method of claim 17, wherein thesubject undergoes the Whipple procedure before, after, or during thecombination metakaryocidal therapy.
 19. The method of claim 1, whereinthe plasma concentration of each metakaryocidal agent in the combinationtherapy is maintained substantially constant.
 20. The method of claim 1,wherein there is a resting interval between the administration of eachseparate metakaryocidal drug.